A compressive review about Taxol® : history and future challenges

©2020. This manuscript version is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Published, version of a Published Work that appeared in final form in Molecules. To access the final edited and published work see https://doi.org/10.3390/molecules25245986Taxol®, which is also known as paclitaxel, is a chemotherapeutic agent widely used to treat different cancers. Since the discovery of its antitumoral activity, Taxol® has been used to treat over one million patients, making it one of the most widely employed antitumoral drugs. Taxol® was the first microtubule targeting agent described in the literature, with its main mechanism of action consisting of the disruption of microtubule dynamics, thus inducing mitotic arrest and cell death. However, secondary mechanisms for achieving apoptosis have also been demonstrated. Despite its wide use, Taxol® has certain disadvantages. The main challenges facing Taxol® are the need to find an environmentally sustainable production method based on the use of microorganisms, increase its bioavailability without exerting adverse effects on the health of patients and minimize the resistance presented by a high percentage of cells treated with paclitaxel. This review details, in a succinct manner, the main aspects of this important drug, from its discovery to the present day. We highlight the main challenges that must be faced in the coming years, in order to increase the effectiveness of Taxol® as an anticancer agent

Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering

Exhaled volatilome analysis as a useful tool to discriminate asthma with other coexisting atopic diseases in women of childbearing age

©2021. The authors. This document is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by /4.0/ This document is published version of a Published Work that appeared in final form in Scientifc Reports. To access the final edited and published work see https://doi.org/10.1038/s41598-021-92933-2The prevalence of asthma is considerably high among women of childbearing age. Most asthmatic women also often have other atopic disorders. Therefore, the diferentiation between patients with atopic diseases without asthma and asthmatics with coexisting diseases is essential to avoid underdiagnosis of asthma and to design strategies to reduce symptom severity and improve quality of life of patients. Hence, we aimed for the frst time to conduct an analysis of volatile organic compounds in exhaled breath of women of childbearing age as a new approach to discriminate between asthmatics with other coexisting atopic diseases and non-asthmatics (with or without atopic diseases), which could be a helpful tool for more accurate asthma detection and monitoring using a noninvasive technique in the near future. In this study, exhaled air samples of 336 women (training set (n= 211) and validation set (n= 125)) were collected and analyzed by thermal desorption coupled with gas chromatography-mass spectrometry. ASCA (ANOVA (analysis of variance) simultaneous component analysis) and LASSO+LS (least absolute shrinkage and selection operator+ logistic regression) were employed for data analysis. Fifteen statistically signifcant models (p-value< 0.05 in permutation tests) that discriminated asthma with other coexisting atopic diseases in women of childbearing age were generated. Acetone, 2-ethyl-1-hexanol and a tetrahydroisoquinoline derivative were selected as discriminants of asthma with other coexisting atopic diseases. In addition, carbon disulfde, a tetrahydroisoquinoline derivative, 2-ethyl-1-hexanol and decane discriminated asthma disease among patients with other atopic disorders. Results of this study indicate that refned metabolomic analysis of exhaled breath allows asthma with other coexisting atopic diseases discrimination in women of reproductive ag

Relative impact of three growth conditions on the Escherichia coli protein acetylome.

Nε-lysine acetylation is a common posttranslational modification observed in Escherichia coli. In the present study, integrative analysis of the proteome and acetylome was performed using label-free quantitative mass spectrometry to analyze the relative influence of three factors affecting growth. The results revealed differences in the proteome, mainly owing to the type of culture medium used (defined or complex). In the acetylome, 7482 unique acetylation sites were identified. Acetylation is directly related to the abundance of proteins, and the level of acetylation in each type of culture is associated with extracellular acetate concentration. Furthermore, most acetylated lysines in the exponential phase remained in the stationary phase without dynamic turnover. Interestingly, unique acetylation sites were detected in proteins whose presence or abundance was linked to the type of culture medium. Finally, the biological function of the acetylation changes was demonstrated for three central metabolic proteins (GapA, Mdh, and AceA)

Dendritic cell deficiencies persist seven months after SARS-CoV-2 infection

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection induces an exacerbated inflammation driven by innate immunity components. Dendritic cells (DCs) play a key role in the defense against viral infections, for instance plasmacytoid DCs (pDCs), have the capacity to produce vast amounts of interferon-alpha (IFN-α). In COVID-19 there is a deficit in DC numbers and IFN-α production, which has been associated with disease severity. In this work, we described that in addition to the DC deficiency, several DC activation and homing markers were altered in acute COVID-19 patients, which were associated with multiple inflammatory markers. Remarkably, previously hospitalized and nonhospitalized patients remained with decreased numbers of CD1c+ myeloid DCs and pDCs seven months after SARS-CoV-2 infection. Moreover, the expression of DC markers such as CD86 and CD4 were only restored in previously nonhospitalized patients, while no restoration of integrin β7 and indoleamine 2,3-dyoxigenase (IDO) levels were observed. These findings contribute to a better understanding of the immunological sequelae of COVID-19

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Escherichia coli acetate metabolism : characterization and regulation

Escherichia coli (E.coli) es una bacteria Gram-negativa que posee una serie de características que la han convertido en el principal modelo procariota en biología y biotecnología. El licopeno es un terpeno de gran interés industrial que posee diversas aplicaciones, aunque hasta el momento no se ha desarrollado un método de producción que pueda competir con la extracción a partir de tomates. A pesar de las muchas ventajas que presenta E. coli a nivel biotecnológico, también presenta ciertas desventajas. La principal de estas desventajas es la excreción de acetato en cultivos suplementados con glucosa como fuente de carbono. Por otro lado, las modificaciones post-traduccionales, como la N-ɛ acetilación de lisinas, son esenciales para comprender la regulación metabólica de todos los organismos. En E. coli, el metabolismo del acetato se encuentra íntimamente ligado a las modificaciones post-traduccionales por acetilación de lisinas a través de las concentraciones de acetil-CoA y acetil-fosfato, y de la regulación de la proteína acetil-CoA sintetasa (Acs). El objetivo principal de esta tesis doctoral fue avanzar en el conocimiento del metabolismo del acetato de E. coli y en su regulación por acetilación/desacetilación de lisinas A continuación se resumen los principales resultados obtenidos: En el capítulo 3 de esta tesis se ha llevado a cabo el desarrollo de un método de sobreproducción y extracción continua de licopeno con un sistema bifásico, empleando cepas de E. coli modificadas genéticamente. De esta manera, se ha conseguido una producción ampliamente superior a la obtenida con los métodos tradicionales. Por último, los resultados obtenidos en este capítulo corroboran el potencial de E. coli en el campo biotecnológico. El capítulo 4 de esta tesis se ha centrado en la caracterización bioquímica y biofísica de la acetiltransferasa PatZ. Se ha caracterizado cinéticamente la acetilación de la proteína Acs por PatZ por primera vez y se ha demostrado la autoacetilación reversible de PatZ La sirtuína CobB, principal desacetilasa de E.coli, ha sido caracterizada bioquímicamente en el capítulo 5 de esta tesis doctoral. Se ha estudiado cinéticamente la desacetilación de la proteína Acs por CobB y su inhibición por nicotinamida, estableciéndose un mecanismo de inhibición no competitivo. El capítulo 6 de esta tesis se centra en el estudio de la etapa de adenilación del mecanismo catalítico de la proteína Acs. Se ha llevado a cabo la caracterización cinética y termodinámica, respecto a la unión de ATP, de cuatro proteínas mutantes y de la proteína Acs nativa, desacetilada y acetilada. Los resultados han demostrado la importancia de los residuos estudiados en la capacidad catalítica y en la afinidad de Acs por ATP. En el capítulo 7 se ha estudiado la influencia del tipo de fuente de carbono y nitrógeno sobre distintos aspectos relacionados con el metabolismo del acetato en E. coli. Se han observado importantes diferencias en algunos de los parámetros fisiológicos determinados, así como en la excreción de acetato, y se ha determinado, por primera vez, el pH intracelular de forma continua para una bacteria en un cultivo. Finalmente, las principales conclusiones que podemos extraer de esta tesis doctoral son: 1) Se ha construido un sistema competitivo de producción de licopeno empleando cepas de E. coli modificadas genéticamente; 2) El estado oligomérico de PatZ está regulado por autoacetilación/desacetilación; 3) Acs es desacetilada por CobB en varias lisinas con una cinética monofásica; 4) La lisina 609 de Acs tiene un papel fundamental en la unión de la proteína a ATP y 5) La fuente de carbono y nitrógeno tiene una gran influencia en la fisiología y el control del pH intracelular de E. coli. Escherichia coli (E. coli) is a Gram-negative bacterium with several characteristics that have made it a global model in biology and biotechnology. Lycopene is an important terpenoid with several applications, although most of lycopene is obtained from tomato. In spite of the great advantages of E. coli in the biotechnology field, it has also some disadvantages. The main E. coli biotechnological drawback is the excretion of acetate in cultures supplemented with glucose as carbon source. For this, much effort has been put into deepening our knowledge of the acetate metabolism with the purpose of minimizing E. coli acetate overflow. By other way, post-translational modifications, such as N-ɛ lysine acetylation, are essential to know metabolism regulation in all the organisms. E. coli acetate metabolism is closely related to post-translational modifications by lysine acetylation through acetyl-phosphate and acetyl-CoA pools and acetyl-CoA synthetase (Acs) regulation by lysine acetylation. The main objective of this PhD thesis was to advance into the E. coli acetate metabolism knowledge and regulation by lysine acetylation/deacetylation. The main results of this PhD thesis are summarized below. In chapter 3 of this thesis a lycopene overproduction and continuous biphasic extraction system employing E. coli strains modified by genetic engineering has been developed. The results obtained in this chapter corroborate the potential role of E. coli in biotechnology field. Chapter 4 of this dissertation is focused on acetyltransferase PatZ biochemical characterization. The kinetics of Acs acetylation by PatZ have been characterized for the first time, and PatZ reversible autoacetylation has been described. In chapter 5, CobB sirtuin kinetics of Acs deacetylation have been characterized. Moreover, CobB inhibition by nicotinamide has been studied and a non-competitive inhibition mechanism was established. The chapter 6 of this dissertation is focused on the adenylating catalytic step of Acs protein. Kinetics and ATP binding calorimetric parameters were determined for the four mutant proteins and for Acs native protein, in the acetylated and deacetylated state. The results showed the important role of the selected residues in the catalytic and affinity Acs parameters. In chapter 7 of this thesis, the influence of carbon and nitrogen sources on E. coli acetate metabolism was evaluated. The E. coli acetate metabolism was studied for E. coli wt and five deficient strains. As regards physiological parameters and acetate excretion determined, several differences were observed depending on the culture medium and strains. Intracellular pH was determined for the first time in E. coli and important results were achieved. Finally, the main conclusions we can extract are: 1) A competitive lycopene production system using E. coli strains modified by genetic engineering has been developed; 2) PatZ oligomeric state is regulated by autoacetylation/deacetylation; 3) Acs is deacetylated by CobB in several lysines with monophasic kinetics; 4) Acs lysine 609 has an important role in Acs ATP-binding and 5) Carbon and nitrogen sources have a great influence on E. coli physiology and intracellular pH control

Continuous intracellular pH measurement: Escherichia coli culture medium pH dependence

E. coli is the most used host microorganism to high-valuable compounds production in current industry. Cells pH homeostasis is a crucial factor to grow in different environment maintaining an intracellular pH control, however, to our knowledge, there is not a study about E. coli continuous intracellular pH growing in a culture. In this study the intracellular E. coli pH has been determined for cultures growing with glucose or glycerol as carbon sources. To determine intracellular E. coli pH, a novel, simple and accurate method based on pGFPR01 plasmid (kindly provided by Dr. Joan Slonczewski) was employed (1). This vector expressed ratiometric pHluorin. Thus, fluorescence ratio 410/470 showed a perfect lineal dependence with intracellular pH between 5.5 and 9. The results showed a surprising high intracellular pH for E. coli glucose cultures, which raised values of 8.5-9 at exponential growth phase. However, glycerol cultures showed a major regulation of intracellular pH, keeping the values at 6.5-7.5 in exponential and stationary stages. These differences show the dependence between the intracellular pH and the carbon source. In addition, the measurement method developed is a very useful tool for the control of bioprocesses